Recent studies by us and others have presented important criteria for distinguishing nuclear ribosomes from cytoplasmic ribosomes and techniques to study nuclear protein synthesis independently of cytoplasmic synthesis. Applying these criteria and using these techniques, the apparatus and the mechanism of nuclear protein synthesis in adenovirus-infected HeLa cells will be investigated in a cell-free system. Furthermore, the extent of nuclear synthesis and the nature of nuclear products will be compared in HeLa cells, noninfected and adenovirus-infected; mouse L-cells; and Ehrlich ascites tumor cells. Nuclear ribosomes, released by incubation of detergent-washed nuclei with a polyanion poly(U), will be purified by sedimentation and incubated for cell-free protein synthesis in an S-100 extract (lacking ribosomes) from Ehrlich ascites tumor cells. To investigate the apparatus, adenovirus core protein VII and its precursor P-VII synthesis on nuclear and cytoplasmic ribosomes will be analyzed by immunochemical techniques and SDS-PAGE. The amount of P-VII and VII synthesis will be correlated with the mRNA content of the ribosomes by DNA-RNA hybridization. In another type of experiment, messenger and nonmessenger RNA and structural proteins of the two ribosomes will be compared. Various techniques such as hybridization, 5'-terminal cap and the ribosome binding site analysis, 3'-terminus labeling, SDS-PAGE, two-dimensional gel electrophoresis, etc. will be used for this study. To investigate the mechanism, preinitiated nascent peptides present in nuclear ribosomes will be analyzed for the presence of methionine at the NH2-terminus by Edman degradation. Nuclear and cytoplasmic ribosomes in the different cell lines will be quantitated and subsequently incubated for cell-free protein synthesis to determine the rate and to estimate the proportion of each synthesis. The products will be compared by the gel techniques to ascertain the total number of proteins synthesized and to identify the synthesis of any new protein(s) by nuclear ribosomes.